Roy G. Thompson

Differential distribution of norepinephrine and serotonin along the dorsal-ventral axis of the hippocampal formation.

Norepinephrine (NE) and Serotonin (5-HT) are differentially distributed along the dorsal-ventral axis of the Hippocampal Formation (HF). A kinetic analysis of the uptake of labeled NE and 5-HT was performed to assess the relative density of nerve terminals which accumulate NE and 5-HT (Vmax) and the apparent affinity for uptake (Km). The results reveal a greater density of NE and 5-HT terminals in the ventral HF and a higher Km for 5-HT uptake in the dorsal HF. The results are discussed in terms of potential explanation of reported differences following independent manipulation of the dorsal and ventral hippocampal formation.

Endogenous norepinephrine and serotonin within the hippocampal formation during the development and recovery from septal hyperractivity

Fluorometric analysis of serotonin (5-HT) and norepinephrine (NE) content of the hippocampal formation revealed that biogenic amines are distributed heterogeneously in the dorsoventral axis, and that NE also exhibits a heterogeneous distribution in the medial-lateral direction while 5-HT does not. Dissection of the hippocampus into its dorsal and ventral halves shows that both NE and 5-HT exhibit higher concentrations in the ventral hippocampus in comparison to its dorsal counterpart. A dissection which separated the cell fields CA 1 and 2 from CA 3 and 4 and the dentate gyrus showed NE to be the highest in the latter region, while 5-HT was uniformly distributed between the two regions. Taken together, these data indicate that NE is more highly concentrated in the CA 3 and 4 and dentate area of the ventral hippocampus while 5-HT concentration differences are apparent only in a dorsal-ventral dissection. Concentrations of NE and 5-HT in the dorsal and ventral hippocampus were also determined at 1, 3, 6, 11, 16, 24, and 30 days following a lesion to the septal nuclei. The results demonstrate that biogenic amine levels in the dorsal hippocampus achieve maximal depletion earlier than do their ventral counterparts, and that percent depletion is greater for 5-HT than NE in both dorsal and ventral areas. On the first day following septal lesions, 5-HT is increased above normal levels. Sixteen days after septal lesion, 5-HT is substantially depleted below normal levels. In addition, by 30 days, 5-HT shows significant return toward normal levels from its earlier depleted state. Behavioral changes related to sensory reactivity correlate with the relative decreases of NE and 5-HT following septal lesions.

A Fiber-Optic Immunosensor for Detecting Parathion

Abstract Casein-parathion conjugates, immobilized by adsorption on quartz fibers, selectively adsorbed antiparathion rabbit antibodies raised against bovine serum albumin (BSA)-parathion conjugates from polyclonal immune sera. The presence of free parathion interfered with binding of the rabbits antiparathion Abs. Fluorescein isothiocyanate(FITC) goat antirabbit IgG were used to generate an optical signal, which was measured by a signal transducer based on total internal reflection fluorescence. Inhibition of binding of rabbit Abs by free parathion, reduced the optical signal and provided the basis for detection of parathion with this optical immununosensor. It could detect 0.3 ppb parathion and was highly specific since its detection of the oxygen analog paraoxon was possible at 100-fold higher concentration.

A simple and rapid fluorescence-based immunoassay for the detection of staphylococcal enterotoxin B.

The bioterrorism threat is perceived to be a real challenge to our nations security. This threat has necessitated the design of better and faster assays for the detection of biothreat agents including staphylococcal enterotoxin B (SEB), a causative agent of food poisoning. This study describes a simple, fast and highly sensitive fluorescence-based immunoassay, in which the antibody is fluorescently-labeled for use in this assay. Use of labeled antibodies resulted in very low level of detection of SEB, 100 pg/well. This method is four times faster than classical and conventional enzyme-linked immunosorbent assay (ELISA).

Molecular imprinting of Ricin and its A and B chains to organic silanes: Fluorescence detection

Abstract The stable physical properties of molecular imprints make them ideal artificial receptors, i.e., biosensor sensing elements, for detection systems against chemical and biological toxins, drugs, and environmental contaminants [B. Dave, B. Dunn, J. Selverstone Valentine, J. Zink, Anal. Chem. 66 (1994) 1120–1127; O. Lev, M. Tsionsky, L. Rabinovich, V. Glezer, S. Sampath, I. Pankratov, J. Gun, Anal. Chem. 67 (1995) 22–30; R. Wang, U. Narang, P. Prasad, F. Bright, Anal. Chem. 65 (1993) 2671–2675; S.A. Piletsky, Y.P. Parhometz, N.V. Lavryk, T.L. Panasyuk, A.V. Elskaya, Sens. Actuators, B 18–19 (1994) 629–631; M.P. Byfield, R.A. Abuknesha, Biosens. Bioelectron. 9 (1994) 373–400] and afford them advantages over traditional screening techniques such as purified receptors and antibodies which require elaborate preparative techniques and complex environments. Molecular imprints to the deadly castor bean lectin Ricin (Ricinus communis, Toxin RCA60) and its ‘A’ and ‘B’ chains were prepared, and their respective binding constants determined using steady-state fluorescence. Stern–Volmer fluorescence quenching plots using iodide and acrylamide suggest imprint associated Ricin B chain tryptophans are more accessible to the solvent environment than those of the Ricin A chain. Scatchard analysis revealed two affinities for Ricin binding to Ricin imprints, i.e., Kd=34 nM and 319 nM. Similarly, high affinity interaction of Ricin A and B chains with their respective imprints were observed (Kd≅100 nM). Interestingly, Scatchard analysis of Ricin B chain binding to Ricin imprints revealed two apparent affinities (Kd=0.23 and 25 nM) whereas, Ricin B chain binding to A chain imprints exhibited one high affinity constant (Kd=8.9 nM). These data support the usefulness of intrinsic fluorescence and molecular imprints for detection of large proteins and biological toxins.

Pharmacological specificity of a nicotinic acetylcholine receptor optical sensor

The pharmacological specificity of a nicotinic acetylcholine receptor (nAChR) optical biosensor was investigated using three fluorescein isothiocyanate (FITC)-tagged neurotoxic peptides that vary in the reversibility of their receptor inhibition: alpha-bungarotoxin (alpha-BGT), alpha-Naja toxin (alpha-NT), and alpha-conotoxin (GI) (alpha-CNTX). Kinetic analysis of the time course of binding of FITC-neurotoxins to the nAChR-coated fiber gave association rate constants (k+1) of 8.4 x 10(6) M-1 min-1 for FITC-alpha-BGT, 6.0 x 10(6) M-1 min-1 for FITC-alpha-NT and 1.4 x 10(6) M-1 min-1 for FITC-alpha-CNTX. The dissociation rate constants (k-1) for the three neurotoxins were 7.9 x 10(-3) min-1. 4.8 x 10(-2) min-1 and 8.0 x 10(-1) min-1 for FITC-alpha-BGT. FITC-alpha-NT and FITC-alpha-CNTX, respectively. The equilibrium dissociation constant (Kd) values for the three toxins. calculated from these rare constants, were similar to published values obtained from tissue responses or ligand binding assays. The optical signal generated by FITC-alpha-NT binding to the nAChR-coated fiber was effectively quenched by agonists and antagonists of the nAChR but not by most of the tested agonists and antagonists of muscarinic cholinergic, adrenergic, glutamatergic, serotonergic, dopaminergic or GABAergic receptors. Interestingly, 5-hydroxy-tryptamine, haloperidol and (+)cis-methyldioxolane gave significant inhibition of FITC-alpha-NT binding to the immobilized receptor. Equilibrium constants of inhibition (Ki) for d-tubocurarine (d-TC) and carbamylcholine (carb) were determined from competition studies using FITC-alpha-CNTX. FITC-alpha-NT or FITC-alpha-BGT as probes for receptor occupancy. When the more reversible probe FITC-alpha-CNTX was used, the Ki value for d-TC was an order of magnitude lower than those determined using the less reversible probes. Ki values for carb however, were independent of the FITC-toxin probe used.

Comparison of lindane, bicyclophosphate and picrotoxin binding to the putative chloride channel sites in rat brain and Torpedo electric organ

The relative potencies of lindane, picrotoxin and several bicyclophosphate derivatives were compared in their ability to compete with 35S-t-butylbicyclophosphorothionate (35S-TBPS) binding sites in membranes derived from Torpedo electric organ and rat brain. Lindane proved to be ten times more potent in competing with 35S-TBPS binding in electric organ than rat brain, while the bicyclophosphate analogs displayed up to three orders of magnitude greater affinity for rat brain over electric organ. GABA inhibited 35S-TBPS binding in rat brain with moderate potency (IC50 = 30 microM), while unlabelled TBPS inhibited the binding of 3H-muscimol to the GABA receptor with an IC50 greater than 100 microM. The GABA receptor antagonist bicuculline increased 35S-TBPS binding in rat brain both in the presence and absence of 30 microM GABA. The results of the study are discussed in the context of a pharmacological discrimination between voltage-sensitive and receptor-gated Cl- channels in nervous tissue, with lindane and the i-propylbicyclophosphate derivative being the most selective compounds for discriminating between them.

Molecular Imprinting of Small Molecules with Organic Silanes: Fluorescence Detection

Abstract Small molecules, peptides, and proteins can be imprinted using mixtures of organic silanes. Molecular imprints may serve as artificial receptors, i.e., biosensor sensing elements for detection of chemical and biological toxins, drugs, and environmental hazards. One method for detection of imprint-bound molecules is fluorescence. Molecular imprints to N-acetyltryptophanamide (NATA) and fluorescein were prepared, and their respective binding constants determined using steady-state fluorescence spectroscopy. Stern-Volmer fluorescence quenching plots of imprint-bound molecules using potassium iodide (KI) and acrylamide indicate bound molecules are shielded from the solvent environment. Scatchard plot analysis revealed two binding affinities, i.e., aKd = 0.13 and 2.5 μM for NATA binding to NATA imprints. Interestingly, NATA exhibited higher affinity, i.e., Kd = 1.3 and 35 nM, for the fluorescein imprint. These data support the usefulness of fluorescence techniques in molecular imprint-based detection ...

Behavioral kinetics: A method for deriving qualitative and quantitative changes in sensory responsiveness following septal nuclei damage

Abstract An apparatus was designed and constructed that would maximize the reliable and objective recording of a rats response to footshock following damage to the septal area. A method of data analysis was borrowed from enzyme kinetics to provide two summary measures of the rats response to increasing shock levels. These results were then compared with data obtained from other measures of reactivity to determine the validity and generalizability of the new summary measures. The results indicate that the apparatus and data reduction method provide two different sources of information about a rats response to footshock: (1) a statistic representing the qualitative relation between response magnitude and stimulus intensity, and (2) maximal responsiveness, or quantitative index of responsiveness independent of stimulus intensity. The results are discussed in light of the procedures future utility in comparing certain behavioral events with their physiological correlates following surgical or pharmacological intervention.

Detection of nicotinic receptor ligands with a light addressable potentiometric sensor.

The nicotinic acetylcholine receptor, purified from Torpedo electric organ, was coupled to a light addressable potentiometric sensor (LAPS) to form a LAPS-receptor biosensor. Receptor-ligand complexes containing biotin and urease were captured on a biotinylated nitrocellulose membrane via a streptavidin bridge and detected with a silicon-based sensor. Competition between biotinylated alpha-bungarotoxin and nonbiotinylated ligands formed the basis of this assay. This biosensor detected both agonists (acetylcholine, carbamylcholine, succinylcholine, suberyldicholine, and nicotine) and competitive antagonists (d-tubocurarine, alpha-bungarotoxin, and alpha-Naja toxin) of the receptor with affinities comparable to those obtained using radioactive ligand binding assays. Consistent with agonist-induced desensitization of the receptor, the LAPS-receptor biosensor reported a time-dependent increase in affinity for the agonist carbamylcholine as expected, but not for the antagonists.